Expression Of Ceramide Kinase Research Articles

Identification of Androgen Receptor Metabolic Correlome Reveals the Repression of Ceramide Kinase by Androgens.

Simple SummaryProstate cancer cells require androgens to survive and grow. In turn, targeting androgen signaling has become a predominant therapeutic strategy in this disease. These hormones regulate a plethora of biological processes, which identification could aid the refinement of future anticancer treatments. Our aim was to uncover metabolic processes under the control of androgens, taking advantage of bioinformatics analyses using publicly accessible data in prostate cancer. We found that these hormones control the abundance of an enzyme, ceramide kinase (CERK). CERK produces ceramide-1-phosphate, a metabolite with prosurvival and migration properties. This finding suggests that antiandrogen therapies could be limited by the reactivation of this metabolic process. Prostate cancer (PCa) is one of the most prevalent cancers in men. Androgen receptor signaling plays a major role in this disease, and androgen deprivation therapy is a common therapeutic strategy in recurrent disease. Sphingolipid metabolism plays a central role in cell death, survival, and therapy resistance in cancer. Ceramide kinase (CERK) catalyzes the phosphorylation of ceramide to ceramide 1-phosphate, which regulates various cellular functions including cell growth and migration. Here we show that activated androgen receptor (AR) is a repressor of CERK expression. We undertook a bioinformatics strategy using PCa transcriptomics datasets to ascertain the metabolic alterations associated with AR activity. CERK was among the most prominent negatively correlated genes in our analysis. Interestingly, we demonstrated through various experimental approaches that activated AR reduces the mRNA expression of CERK: (i) expression of CERK is predominant in cell lines with low or negative AR activity; (ii) AR agonist and antagonist repress and induce CERK mRNA expression, respectively; (iii) orchiectomy in wildtype mice or mice with PCa (harboring prostate-specific Pten deletion) results in elevated Cerk mRNA levels in prostate tissue. Mechanistically, we found that AR represses CERK through interaction with its regulatory elements and that the transcriptional repressor EZH2 contributes to this process. In summary, we identify a repressive mode of AR that influences the expression of CERK in PCa.

Ceramide kinase regulates TNF-\u03b1-induced immune responses in human monocytic cells

Ceramide kinase (CERK) phosphorylates ceramide to produce ceramide-1-phosphate (C1P), which is involved in the development of metabolic inflammation. TNF-α modulates inflammatory responses in monocytes associated with various inflammatory disorders; however, the underlying mechanisms remain not fully understood. Here, we investigated the role of CERK in TNF-α-induced inflammatory responses in monocytes. Our results show that disruption of CERK activity in monocytes, either by chemical inhibitor NVP-231 or by small interfering RNA (siRNA), results in the defective expression of inflammatory markers including CD11c, CD11b and HLA-DR in response to TNF-α. Our data show that TNF-α upregulates ceramide phosphorylation. Inhibition of CERK in monocytes significantly reduced the secretion of IL-1β and MCP-1. Similar results were observed in CERK-downregulated cells. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was reduced by inhibition of CERK. Additionally, NF-κB/AP-1 activity was suppressed by the inhibition of CERK. Clinically, obese individuals had higher levels of CERK expression in PBMCs compared to lean individuals, which correlated with their TNF-α levels. Taken together, these results suggest that CERK plays a key role in regulating inflammatory responses in human monocytes during TNF-α stimulation. CERK may be a relevant target for developing novel therapies for chronic inflammatory diseases.

Lipidomics Revealed Alteration of Sphingolipid Metabolism During the Reparative Phase After Myocardial Infarction Injury

Aberrant sphingolipid metabolism contributes to cardiac pathophysiology. Emerging evidence found that an increased level of ceramide during the inflammatory phase of post-myocardial infarction (MI) served as a biomarker and was associated with cardiac dysfunction. However, the alternation of the sphingolipid profile during the reparative phase after MI is still not fully understood. Using a mouse model of the left anterior descending ligation that leads to MI, we performed metabolomics studies to assess the alternations of both plasma and myocardial sphingolipid profiles during the reparative phase post-MI. A total number of 193 sphingolipid metabolites were detected. Myocardial sphingolipids but not plasma sphingolipids showed marked change after MI injury. Ceramide-1-phosphates, which were accumulated after MI, contributed highly to the difference in sphingolipid profiles between groups. Consistently, the expression of ceramide kinase, which phosphorylates ceramides to generate ceramide-1-phosphates, was upregulated in heart tissue after MI injury. Our findings revealed the altering sphingolipid metabolism during the reparative phase post-MI and highlighted the potential role of ceramide kinase/ceramide-1-phosphate in ischemic heart disease.

Regulation of the amount of ceramide-1-phosphate synthesized in differentiated human podocytes

Sphingolipids have important functions as structural components of cells but they also function as signaling molecules regulating different cellular processes such as apoptosis, cell proliferation, cell migration, cell division and inflammation. Hence, a tight regulation of the sphingolipid homeostasis is essential to maintain proper cellular functions. Mammalian ORMDL proteins are orthologues of the yeast ORM1/2 proteins, which regulate ceramide synthesis in yeast. ORMDL proteins inhibit serine palmitoyltransferase (SPT), the enzyme regulating a rate-limiting step of the sphingolipid pathway to control the levels of ceramides and other sphingolipids. Sphingomyelinase phosphodiesterase like 3b (SMPDL3b) is a glycosylphosphatidylinositol (GPI) anchored protein in the plasma membrane (PM) and determines membrane fluidity in macrophages. We previously showed that differential expression of SMPDL3b alters the availability of Ceramide-1-phosphate (C1P) in human podocytes, which are terminally differentiated cells of the kidney filtration barrier. This observation lead us to investigate if SMPDL3b controls C1P availability in human podocytes by interfering with ceramide kinase (CERK) expression and function. We found that SMPDL3b interacts with CERK and can bind to C1P in vitro. Furthermore, CERK expression is reduced when SMPDL3b expression is silenced. These observations led us to propose that one of the mechanisms by which SMPDL3b influences the amount of C1P available in the podocytes is by interfering with the function of CERK thereby maintaining a balance in the levels of the C1P in podocytes.

Oleate increase neutral lipid accumulation, cellular respiration and rescues palmitate-exposed GLP-1 secreting cells by reducing ceramide-induced ROS

BackgroundFatty acids (FAs), and especially monounsaturated FAs (MUFAs) stimulate GLP-1 release. However, lipotoxicity is indicated in GLP-1 secreting cells following long-term exposure to elevated levels of saturated FAs (SFAs) in vivo and in vitro, where in vitro studies indicate that cosupplementation with MUFAs confers lipoprotection. SFAs and MUFAs differentially affect the fate of cells in ways that depend on the cell type, concentration and ratio of the FAs. The present study was designed to further elucidate the mechanisms underlying the effects of SFAs/MUFAs on GLP-1-producing cells in terms of lipotoxicity/lipoprotection and GLP-1 secretion. MethodsCultured GLP-1 secreting cells were exposed to hyperlipidemia simulated by SFA-albumin complexes where the molar ratio was 2:1. The cellular response to simulated hyperlipidemia was assessed in the presence/absence of MUFA cosupplementation by determining intracellular ceramide, ROS, neutral lipid accumulation, and cellular respiration. The role for cellular respiration in GLP-1 secretion in response to SFAs/MUFAs was assessed. ResultsGeneration of intracellular ceramide mediate a detrimental increased in ROS production following long term exposure to SFAs in GLP-1-secreting cells. Cosupplementation with MUFAs increases cellular respiration, triglyceride synthesis, and the expression of ceramide kinase, while reducing ceramide synthesis and attenuating ROS production, caspase-3 activity and DNA fragmentation. Further, acute secretory effects of unsaturated FAs are independent of FAO, but mediated by a FFAR1 induced increase in cellular respiration. ConclusionThis study demonstrates novel data supporting effects of MUFAs on the ceramide biosynthetic pathway, triglyceride storage respiration and secretion in GLP-1 secreting cells. These findings may be of value for nutritional interventions, as well as for identification of novel targets, to help preserve L-cell mass and potentiate GLP-1 secretion in diabesity.

Effect of Eriodictyol on the Development of Atopic Dermatitis-Like Lesions in ICR Mice

Atopic dermatitis (AD) is a chronic, allergic, and inflammatory skin disease associated with eczema and dermatitis symptoms. Our previous studies have reported that eriodictyol extract inhibits immunoglobulin E (IgE)/Ag-induced type I hypersensitivity by suppressing the activation of proinflammatory cytokines, such as interleukin-4 (IL-4), and the expression of ceramide kinase. In this study, we investigated the inhibitory effect of eriodictyol on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions in ICR mice. Treatment with 2 mg/mL eriodictyol for DNCB-induced AD-like skin lesions in ICR mice improved scratching behavior and skin severity score. Histological analysis demonstrated that thickening of the skin lesions were significantly reduced in the eriodictyol-treated group. Also, eriodictyol suppressed the DNCB-mediated elevation of IgE serum levels. These results suggest that eriodictyol may be a potential therapeutic resource for AD and an adjunctive agent to control itchiness in AD.

Expression of Ceramide Kinase (Cerk) and Related Regulation in Early Pregnant Uterus of Mice

Objective To investigate the expression of ceramide kinase (Cerk) and related regulation in mouse uterus during early pregnancy. Methods Several mouse models, including early pregnancy, pseudopregnancy, delayed or activated implantation, artificial decidualization, and steroid hormonal treatment were performed (n=10). Immunohistochemistry and in situ hybridization were used to detect the expression of Cerk protein and mRNA in uterus. Results Expression of Cerk mRNA and protein was strongly detected in the luminal and glandular epithelium on day 1 of pregnancy. However, Cerk mRNA and protein signals were strongly detected in the subluminal stroma surrounding the implanting blastocyst on day 5 and decidua from day 6 to day 8 whereas not in the luminal epithelium. The expression of Cerk in luminal and glandular epithelium of pseudopregnancy was similar to that of early pregnancy from day 1 to day 4 whereas on day 5 of pseudoprgenancy still with remarkable signals in the luminal epithelium. Under delayed implantation, no obvious Cerk expression was detected in the uterus. After delayed implantation was terminated by estrogen treatment and embryo implantation was initiated, both Cerk mRNA and protein were detected in the subluminal stroma surrounding the implanted blastocyst. A strong Cerk signal was detected in decidualized cells under artificial decidualization, whereas only a basal level of Cerk signal was observed in the control uterus which did not inject sesame oil. Progesterone induced a slight expression of Cerk in the luminal and glandular epithelium. Both estradiol and a combination of progesterone with estradiol strongly increased the level of Cerk signal in the luminal and glandular epithelium. Conclusion Cerk expression is under the regulation of progesterone and estrogen. The strong expression of Cerk in implantation site and decidua suggests that Cerk might play an important role during implantation and decidualization.

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [ 3H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.

The cannabinoid R(+)methanandamide induces IL-6 secretion by prostate cancer PC3 cells

In the present study, we have investigated the effect of the cannabinoid R(+)methanandamide (MET) in the androgen-resistant prostate cancer PC3 cells. MET induced a dose-dependent decrease in PC3 cell viability as well as a dose-dependent increase in the secretion of the cytokine IL-6. Looking deeper into the mechanisms involved, we found that MET-induced de novo synthesis of the lipid mediator ceramide that was blocked by the ceramide synthase inhibitor Fumonisin B1. Pre-incubation of cells with the cannabinoid receptor CB2 antagonist SR 144528 (SR2), but not the CB1 antagonist Rimonabant or the TRPV1 antagonist capsazepine, partially prevented the anti-proliferative effect, the ceramide accumulation, and the IL-6-induced secretion, suggesting a CB2 receptor-dependent mechanism. Fumonisin B1 did not have any effect in the IL-6 secretion increase induced by MET. However, even an incomplete down-regulation of (i.e., not a total silencing of) ceramide kinase expression by specific siRNA prevented the MET-induced IL-6 secretion. These results suggest that MET regulates ceramide metabolism in prostate PC3 cells which is involved in cell death as well as in IL-6 secretion. Our findings also suggest that CB2 agonists may offer a novel approach in the treatment of prostate cancer by decreasing cancer epithelial cell proliferation. However, the interaction of prostate cancer cells with their surrounding, and in particular with the immune system in vivo, needs to be further explored.

Gene expression of ceramide kinase, galactosyl ceramide synthase and ganglioside GD3 synthase is associated with prognosis in breast cancer

Sphingolipids are bioactive lipids implicated in apoptosis, cell survival and proliferation. We analyzed the prognostic value of enzymes from sphingolipid metabolism in breast cancer. Differences in expression of ceramide galactosyl transferase (UGT8), ceramide kinase (CERK), and Ganglioside GD3-Synthase (ST8SIA1) in breast cancer cells were investigated by using microarray data of 1,581 tumor samples. UGT8, CERK, and ST8SIA1 were associated with poor pathohistological grading (P < 0.001). High CERK expression was correlated with ErbB2 status (P = 0.006). Among ER positive breast cancers a significant worse prognosis for patients with tumors showing low ST8SIA1 and UGT8 expression was observed. In the ER negative subgroup those samples with high CERK expression displayed a worse prognosis. In a multivariate analysis only ST8SIA1 and tumor size remained significant. Our experiments reveal that expression of enzymes from the sphingolipid metabolism has prognostic implications in breast cancer.

Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A 2α; additional role of ceramide-1-phosphate in phosphorylation and Ca 2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A 2 (PLA 2) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca 2+ binding regions in the C2 domain of α type cytosolic PLA 2 (cPLA 2α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA 2α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D- erythro-2-( N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca 2+ ionophore (A23187) -induced release of AA via cPLA 2α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca 2+ nor the phosphorylation of cPLA 2α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA 2α that is independent of the Ca 2+ signal and the PKC-ERK-mediated phosphorylation signal.

Ceramide kinase mediates toll‐like receptor 4‐induced inflammation in vascular smooth muscle cells

Sphingolipid metabolites have recently been implicated in toll‐like receptor 4 (TLR4) signaling. Activation of lipopolysaccharide (LPS)‐induced TLR4 signaling is dependent upon production of ceramide by acid sphingomyelinase (Cuschieri et al, Surg Infect, 2007). Yet, the role of ceramide metabolites to contribute to TLR4‐induced inflammatory responses has not been investigated. Specifically, we now hypothesize that TLR4‐induced proliferation and inflammation in vascular smooth muscle cells (VSMC) is dependent upon activated ceramide kinase (CERK) and resulting production of ceramide‐1‐phosphate (C1P). We demonstrate that LPS treatment of A7r5 VSMC leads to elevated CERK mRNA and protein expression. This increased expression of CERK correlates with increased LPS‐induced C1P mass, quantified by LC/MS/MS analysis. We show that LPS‐ and ceramide‐1‐phosphate‐, but not ceramide‐treatment, leads to hallmarks of vascular inflammatory disease, including increased VSMC proliferation, positive remodeling, and production of inflammatory mediators. In addition, LPS‐treated mouse blood samples had increased levels of ceramide‐1‐phosphate at the expense of ceramide. Taken together, our studies implicate ceramide‐1‐phosphate as a potential biomarker and /or mediator of vascular inflammation in diseases such as atherosclerosis or restenosis after angioplasty. These studies were funded, in part, by NIH RO1 HL076789 to MK.

Ceramide kinase expression is altered during macrophage-like cell differentiation of the leukemia cell line HL-60

Ceramide kinase (CerK) has important roles in leukocyte functions, including the role in degranulation of mast cells and the phagocytosis of polymorphonuclear leukocytes, so its expression levels should be strictly regulated. Here, we report that the mRNA expression and enzyme activity of CerK were decreased during macrophage-like cell differentiation of the leukemia cell line HL-60, yet neither was altered during granulocytic differentiation of the same cells. Our findings demonstrate that HL-60 cells are useful for studying CerK functions in leukocyte differentiation, and they also suggest that CerK might have an important role in such differentiation.

Synergistic effect of TNF-α and ceramide on the production of IL-8 in A549 cells and A549 cells transfected with human ceramide kinase (CERK). (90.11)

Abstract Many inducers of the stress response result in ceramide (Cer) accumulation as a result of activation of either sphingolyelinases (SMases) or the de novo pathway. Recently, there is strong evidence for a role of sphingolipids in inflammatory responses such as leukocyte recruitment. The attraction of leukocytes to tissues is essential for inflammation and the host response to infection. Following stimulation by TNF-?, the production of IL-8 increased over time in a concentration dependent manner. Simultaneously, Cer levels increased gradually from 7.56 % during 15 minute stimulation to 35% for 16 hour stimulation. Using Fumonisin B1, an inhibitor of de novo synthesis of Cer, which inhibits Cer synthesis by 25%, we observed 17% decrease of IL-8 expression. When the inhibitor for neutral (SMase) GW4869 was used on A549 cells, there was a significant reduction of Cer as well as IL-8. Subsequently, two serine-threonine phosphatases (PPas), protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) have been shown to be Cer responsive in vitro and in vivo. These results suggest that TNF-? stimulates the production of IL-8 by a mechanism that involves the sphingomyelin-Cer system. Cer may be important in increasing the production of IL-8 in A 549 cells. Since Cer can be phosphorylated by CERK to C1P, we predict that CERK expression may affect the balance between these sphingolipids and influence the Cer dependent PPas.

Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells

Ceramide-1-phosphate (C1P) is emerging as a new addition to the family of bioactive sphingolipid metabolites. At low concentrations, C1P enhanced survival of NIH 3T3 fibroblasts and A549 lung cancer cells, while at high concentrations, it reduced survival and induced apoptosis. Apoptosis correlated with degradation of C1P to pro-apoptotic ceramide. To examine the role of endogenous C1P, expression of ceramide kinase, the enzyme that produces C1P, was downregulated, which reduced cellular proliferation, progression into S phase and enhanced apoptosis induced by serum starvation. Our results suggest that ceramide kinase determines the balance between pro-apoptotic ceramide and anti-apoptotic C1P to regulate cell fate, reminiscent of its function in plants.

Ceramide Kinase Promotes Store Dependent Ca2+ Signals and Enhances Phagolysosomal Fusion in COS-1 Cells.

We showed that ceramide-1-phosphate (C1P) is formed by ceramide kinase (CERK). In this study the mechanism by which C1P enhanced phagocytosis and phagolysosomal formation in COS-1 cells expressing hCERK was evaluated. We hypothesized that CERK has a unique role in inducing Ca2+ signaling during Fc-mediated phagocytosis via its generation of C1P. Furthermore, we also hypothesize that CERK mediated changes in Ca2+ levels were not direct but required the participation of a store operated channels (SOC). In some systems, evidence suggests that SOC might be related to transient receptor potential channel (TRP) homologues. This hypothesis was tested using cell lines with different levels of expression of CERK and by using series of pharmacological inhibitors. To monitor subcellular localization of hCERK, red fluorescent protein (RFP)-tagged hCERK was created and confocal microscopy was performed. To study dynamic changes in Ca2+ during phagocytosis, imaging microscopy was used. Phagolysosome formation was evaluated by labeling the cells with lysotracker red DND-99 at 37oC and followed by imaging. When challenged with EIgG, hCERK transfected cells increased phagocytosis and simultaneously increased C1P. During subcellular fractionation of hCERK transfected cells, CERK translocates during activation from the cytosol to a lipid raft fraction. We also observed that TRP-1 accumulated at the site of CERK translocation in lipid rafts. Microfluorimetry of indo-1 revealed that Ca2+ oscillations in COS-1 cells, stably transfected with Fcγ RIIA/hCERK, were much higher compared to Fcγ RIIA transfected cells. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. hCERK transfected cells had a mean rate of fusion of 90%, compare to 76% ( catalytic inactive mutant of CERK-G198DhCERK), 65% (Fcγ RIIA), and 70% (Vector) transfected cells. The indo-1 intensity spikes demonstrated significantly higher Ca2+ intensities for phagosomes associated with the hCERK transfectants. The SOC inhibitor SKF96365 blocked enhanced Ca2+ signaling in the hCERK transfected cells. Similarly SOC inhibitors reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. Our data showed that there is a co-localization of EIgG and CERK as well as caveolin-1 and CERK on the surface of the phagocytic cells. Site directed mutagenesis demonstrated that the kinase activity of hCERK was required for higher Ca2+ signals. Pharmacological inhibitors revealed that this Ca2+ signal operates through SOC. Our results show that introduction of the hCERK gene in cells alters the functional behavior of intracellular signals and supports the role of C1P in promoting membrane fusion by modulating Ca2+ signaling through activation of SOC